Dipeptidyl Peptidase 4/Midline‐1 Axis Promotes T Lymphocyte Motility in Atherosclerosis

Abstract T cells play a crucial role in atherosclerosis, with its infiltration preceding the formation of atheroma. However, how T‐cell infiltration is regulated in atherosclerosis remains largely unknown. Here, this work demonstrates that dipeptidyl peptidase‐4 (DPP4) is a novel regulator of T‐cell motility in atherosclerosis. Single‐cell ribonucleic acid (RNA) sequencing and flow cytometry show that CD4+ T cells in atherosclerotic patients display a marked increase of DPP4. Lack of DPP4 in hematopoietic cells or T cells reduces T‐cell infiltration and atherosclerotic plaque volume in atherosclerosis mouse models. Mechanistically, DPP4 deficiency reduces T‐cell motility by suppressing the expression of microtubule associated protein midline‐1 (Mid1) in T cells. Deletion of either DPP4 or Mid1 inhibits chemokine‐induced shape change and motility, while restitution of Mid1 in Dpp4−/− T cell largely restores its migratory ability. Thus, DPP4/Mid1, as a novel regulator of T‐cell motility, may be a potential inflammatory target in atherosclerosis.


Fig. S7 DPP4 expressing T cells in human atherosclerosis plaque:
Human plaque section was stained with anti-human CD3 and anti-human DPP4 antibodies and their corresponding fluorescently labeled secondary antibodies, followed by DAPI staining. Images were captured under a fluorescence microscope.

Fig. S9 DPP4 expression on aorta-infiltrating immune cells in Rag1 -/mice adoptively transferred with wild-type or DPP4 deficient T cell:
Rag1 -/mice lacking lymphocytes were adoptively transferred with T cells isolated from Dpp4 +/+ or Dpp4 -/mice and intravenously injected with proprotein convertase subtilisin/kexin type 9 (PCSK9)overexpressing AAV8 virus, followed by 16 weeks of high fat diet feeding to induce atherosclerosis. Aortic tissue was then isolated and digested using collagenase to prepare single cell suspension, followed by flow cytometric detection of DPP4

Fig. S11 IFNγ and IL-17 expression in aortic plaque lesions of Dpp4 T-∆ and Dpp4 T-WT mice:
Dpp4 T-∆ and Dpp4 T-WT mice infected with PCSK9-expressing AAV were fed a high fat diet for 16 weeks. The aortic sinus sections were stained with anti-mouse DPP4 (red) and anti-mouse IFNγ (green, a) or IL-17 (green, b) and visualized under a fluorescence microscope (400x magnification).

Fig. S13 Expression of chemokine receptor CCR7 on Dpp4 +/+ and Dpp4 -/-T cells:
Dpp4 +/+ and Dpp4 -/lymph node cells were stained with CCR7 and markers for T cell subsets. Flow cytometry showed that DPP4 deficiency did not affect the expression of CCR7, the chemokine receptor for CCL19 and CCL21, suggesting that the reduced migratory activities towards CCL19 and CCL21 in Dpp4 -/mice was independent of chemokine receptor expression. a, Gating strategy; b, Expression of CCR7 on total T cells, CD8 + T cells and CD4 + T cells.

Mid1 -/-T cells:
WT, Dpp4 -/-, and Mid1 -/-T cells were isolated from lymph node and incubated on 2μg/mL mouse ICAM-1 pre-coated slides for 20 min. After gentle washes with 1x PBS, adhered T cells were treated with 0.5 μg/mL CCL19 or PBS for 10 min, followed by staining of F-actin (red) and β-tubulin (green). Representative images showed that CCL19 induced cell shape change and polarization of F-actin/β-tubulin in WT T cells, which were largely suppressed in Dpp4 -/and Mid1 -/-T cells.